RealtimePCR
Classicpolymerase chain reaction
• Low sensitivity • Poor precision • Results are not expressed as numbers • Ethidium bromide staining is not quantitative •Post-PCR processing required •Narrow dynamic range (<2 logs)
Traditional End-Point PCR
Traditional End-Point PCR
Classicpolymerase chain reaction
Copynumber ofPCRtemplate
PCRconditions:32cycles
• has a narrow dynamic range (<2 logs) à SEMI-QUANTITATIVE
Amplification indynamic range:increase intemplate number results anincreasedamountofPCRproduct,visibilebygelelectrophoresis à semi-quantitativeinformation
Amplification outofdynamic range:increaseintemplate number does not result anincreased amount ofPCRproduct,visibilebygelelectrophoresis –à Endpoint arrived already with100template-copiesà Noquantitativeinformation
Templatecopy
numbers
Amou
ntofPCR
produ
ct
PCRCycle number301
Copynumber oforiginalPCRtemplate:
1000300100301031
10and30copies:Different amout
ofamplified product
Saturation
chosen endpoint ofPCR
Backgroundofendpoint PCR
End-point PCR:à ideal togive qualitative information(forexampe amplification ofamutation using specific
primers)à Only limited quantitative informationpossible
Template copynumbertoo low
Traditional End-Point PCR
Amou
ntofPCR
produ
ct
PCRCycle number301
Copynumber oforiginalPCRtemplate:
10003001003010
10and30copies:Different amout
ofamplified product
Saturation
chosen endpoint ofPCR
Get better quantitative informationfromclassic PCR:- Optimize PCRconditions:
A. Testforideal end-point ofPCR(example 25)B. Optimize theamount oforiginal template used forPCR
25
Optimizingquantitive information fromclassic PCR
TimeWasteofprimary materialCostsVariability
Adjust ideal number of cycles
Amou
ntofPCR
produ
ct
PCRCycle number301
Copynumber oforiginalPCRtemplate:
1000
chosen endpoint ofPCR
25
Real-timePCR
Follow theamplification ofPCRamplicons in“REAL-TIME”=REALTIMEPCR
Measureamount ofPCRproductafter eachcompletedPCRcycle
Obtaining QUANTITATIVEinformationfromPCR
Follow PCR product amplification in real-time (RT-PCR)
♦Eliminate use of gel electrophoresis ♦Increase reproducibility♦Enable use of internal controls/standards ♦Reduce turnaround time ♦Increase throughput♦Reduce sample amount usage♦Results expressed as numbers
Real-timePCR
Real-Time PCR Chemistries
Strategies tofollowPCRproduct generation
1.SYBR®GreenIDye Assay Chemistry
ClassicPCRsetupwithaddition ofSYBRGreen:SYBRGreenis agreenfluorescent cyanine dye that has highaffinity fordouble-strandedDNA.Themodeofbinding isbelieved tobeacombination ofDNAintercalation andexternalbinding.When bound,SYBRabsorbs at awavelength around497nmandemits fluorescence around 520nm.
DenaturationAnnealing
DNAsynthesisDetection ofemission offluorescence
Fluorescence emmission is increasing withincreasing ofPCRcycles
www.biorad.com
2a.excitationfilters
2b.emissionfilters
1.halogentungstenlamp
4.sampleplate
3.intensifier5.ccd detector350,000pixels
Basicsofreal-timePCRmeasurements
Every PCRcycle:ExitationofSYBRgreen(497nm)+measurmentofemission fromSYBRgreen(520nm)
Basicsofreal-timePCRmeasurements
02000000004000000006000000008000000001000000000120000000014000000001600000000
0 5 10 15 20 25 30 35PCR CYCLE NUMBER
AM
OU
NT
OF
DN
AMeasuredem
ission
AMPLIFICATIONBLOT
Quantitativeinformation
METLTINGCURVEANALYSISThetemperature-dependent dissociationbetween two DNA-strands canbemeasuredusing aDNA-intercalating fluorophoresuchas SYBRgreen,or fluorophore-labelled DNAprobes.InthecaseofSYBRgreen(whichfluoresces 1000-foldmoreintensely whileintercalated intheminorgroove oftwostrandsofDNA),thedissociation oftheDNAduring heating is measurable bythelargereduction influorescence that results.
Thetemperatureat which 50%ofDNAisdenatured is known as themeltingtemperature.
Temperature
dissociatio
nofdsD
NA
MeltingcurveisdeterminedafterthelastcycleofPCR:à PCRmachineheatsuo PCRproductsfrom0°Cto100°Cà DissociationofDNAfilamentsismeasuredà IFPCRHASAMPLIFIEDSPECIFICALLYAMPLIFIEDASPECIFICREGIONALLDNAMOLECULESWILLMELTATASPECIFIC
TEMPERATUREàmeltingtemperatureisdeterminedbyDNAsequence!!!à IFYOURUNPCRPRODUCTONAGAROSEGEL,ONLYONE BANDWILLBEVISIBLE
Basicsofreal-timePCRmeasurements
Agarose gel
AREAUNDERPEAK=SYBRincorporation
METLTINGCURVEANALYSIS
Basicsofreal-timePCRmeasurements
dissociatio
nofdsD
NA
MeltingcurveisdeterminedafterthelastcycleofPCR:à PCRmachineheatsuo PCRproducts
from0°Cto100°Cà DissociationofDNAfilamentsis
measuredà IFPCRHASAMPLIFIEDMULTIPLE
FRAGMENTSINANON_SPECIFICMANNERTHEMELTINGCURVEANALYSISWILLIDETIFYMORETHENONEPEAK(PCRprimersarenotsepcific!!)
à Example:IFYOURUNPCRPRODUCTONAGAROSEGEL,FIVEBANDsWILLBEVISIBLE
METLTINGCURVEANALYSISGIVEQUALITATIVEINFORMATIONOFTHEREAL-TIMEPCRREACTION(without necessarily requiring anagarose gelrun)
PCRproduct 1
orprimer dimers
PCRproduct 2
Agarose gel
AREAUNDERPEAKs=SYBRincorporation
Here:signals fromdifferent types ofdsRNA(not only targetamplicon)
Real-Time PCR Chemistries
Strategies tofollowPCRproduct generation
PCRtargetregionamplifiedusingprimer1and2
TaqmanprobePrimer 1
Primer 2
2.Real-TimePCRChemistries based onFluorogenic 5’Nuclease assay
ClassicPCRsetupwithaddition ofamplicon-specific,modified ssDNA oligonucleotide
Taqman probe:- Oligonucleotide- PCRamplicon sitespecific- Hybridizes withone strand ofthePCRproduct- Carries afluorophor (R)- Carries aQuencher that absorbes lightemited fromfluorophor
=“FRET”FRET:Fluorescence ResonanceEnergyTransfer Important:FRETonly works when Q is inclose proximity toR
FRET
Forexample:Cycle 5during PCR
- Denaturation at 95°C- Annealing ofPCRprimers andTaqmanprobe
2.Real-TimePCRChemistries based onFluorogenic 5’Nuclease assay
FRET
NOFRET
- DNASYNTHESISBYTaqpolymerase
Taq has 5’à 3’exonucleaseactivity:Taqman probeesdegraded
Loss ofFRET:lightfromR is not chechedandcanbedetected in“real-time”duringPCRFluoresence increases withevery cycle ofPCRuntil reaching saturation inPCRplateauphase
2.Real-TimePCRChemistries based onFluorogenic 5’Nuclease assay
FRET
NOFRET
- DNASYNTHESISBYTaqpolymerase
Taq has 5’à 3’exonucleaseactivity:Taqman probeesdegraded
ADVANTAGE:HIGHLYSPECIFICDETECTIONOFAMPLIFIEDDNAREGIONs:1.Seqeunce specific PCRprimers forPCR2.Amplicon specific DNAprobeenables selective detectionofregionofinterest!!!
2.Real-TimePCRChemistries based onFluorogenic 5’Nuclease assay
Terminology ofamplification blots
2parallel PCRreacationsshown inblot:BLUE:PCRwithtemplateRED:PCRwithout template(negativecontrol)
Rn:reportersignal obtained fromdetector
Terminology ofamplification blots
Terminology ofamplification blots
Terminology ofamplification blots
CtVALUE:Most important value fortheanalysis ofreal-timePCRdata
Ct = threshold cycle: è il ciclodella reazione diamplificazione in cui ilsegnale di fluorescenza delcampione è maggiorerispetto a quello dellaThreshold
Terminology ofamplification blots
Y =N (1+E)n Y = resa di amplificazione/amount amplifidN = numero di molecole di DNA di partenza (number of starting DNA molecules)E = efficienza di reazione (efficieny of reaction)n = numero di cicli di amplificazione (number of PCR cycles)
WITHEVERYCYCLEOFPCR,THEAMOUNTOFAMPLIFIEDDNADOUBLES
Theamount ofinitial DNAis reverseproportional tothenumber ofcicles required toovercomethethreshold (arrive at Ct)
Sample1(dublicate1)Sample1(dublicate2)
Sample2(dublicate1)Sample2(dublicate2)
Sample3(dublicate1)Sample3(dublicate2)
Sample4(dublicate1)Sample4(dublicate2)
Ct Ct
Ct=26,2
Ct=28,0
Ct=28,5
Ct=30,5
Amount ofDNAof
interest indifferentsamples
Threshold
Basics fortheanalysis ofreal-timePCRdata:Ct andΔCt
WITHEVERYCYCLEOFPCR,THEAMOUNTOFAMPLIFIEDDNADOUBLES
Sample1(dublicate1)Sample1(dublicate2)
Sample2(dublicate1)Sample2(dublicate2)
Sample3(dublicate1)Sample3(dublicate2)
Sample4(dublicate1)Sample4(dublicate2)
Ct Ct
Ct=26,2
Ct=28,0
Ct=28,5
Ct=30,5
Amount ofDNAof
interest indifferentsamples
Ct=28,5 Ct=30,5
ΔCt=2
Fold change sample3tosample4=2ΔCt
22=4
Ct (sample4)appears 2cycles later thatCt (sample3)à note:inevery cycle ofPCRtheamout ofamplifiedDNAdoublesà 2cycles difference=concentrationoftargetDNAis 4times lower insample4comparedtosample3
Analysisofreal-timePCRdata:Ct andΔCt
Analysisofreal-timePCRdata:Ct andΔCt
Provides absolute measurement of starting copy number –Requires standards of known quantity (Mol or ng) –e.g. Forensic science: Is there DNA and how much DNA (copy number) is there for forensics purposes- e.g. Diagnostics: Virus titer in blood: is there virus DNA and how much is there?
Basics fortheanalysis ofreal-timePCRdata:Ct andΔCt
Cts derived fromreal-timePCRusing andincreased copynumber oftargetsite:PCRTARGETREGIONMUSTBEAVAILABLEAT(forexample cloned into aplasmid)DIFFERENTDILLUTIONSAREUSEDFORPCRTOGENERATEASTANDARDCURVE
Biological samples withunknowncopynumber ofPCRtargetsite
ABSOLUTEQUANTITATION
Biological sampleCt=21
Defined amounts oftargetsiteused forPCR(standards)
Defined amounts oftargetsiteused forPCR(standards)
Ct ofall standards areused togenerateastandardcurve:y=-2,9966x+15.311
Ct ofBiological sample=2121=-2,9966x+15.311x=-1,89848logng
Basics fortheanalysis ofreal-timePCRdata:Ct andΔCt
ABSOLUTEQUANTITATION
Basics fortheanalysis ofreal-timePCRdata:Ct andΔCt
Provides accurate discrimination between relative amounts of starting material –e.g. Comparing expression levels of wildtype vs. mutated alleles –e.g. Comparing expression levels of a gene across different tissues or between different biological conditions –e.g. Validating array results
RELATIVEQUANTITATION
Basics fortheanalysis ofreal-timePCRdata:relativequantitation
Cells stimulated forseveral hours(0-48h)withretinoic acidQUESTION:Howaregenes ofinterest(forexample Hox Agene)regualtedduring this time
Used prepared cDNA forreal-timePCRtodetermine thelevels ofmRNAs ofinterest indiffernet experimentalsamples
Hox genes
REFERENCEGENETHATISNOTAFFECTEDBYRETINOICACIDTREATMENT
RELATIVEQUANTITATION
Basics fortheanalysis ofreal-timePCRdata:relativequantitation
Cells stimulated forseveral hours(0-48h)withretinoic acid
t=0 Ct Hox Agene=30
Ct reference gene=15
ΔCt
RELATIVEQUANTITATION
REFERENCE GENE:- no altered expression in relevant
biological- normally expressed at high levels- Serves to control of sample quantity- Serves to control for pipetting errors - Examples:18S rRNA, GAPDH, β-
actin, tubilin, RNA polymerase II, histone H3
REALTIMEPCR:performed inparallel (t=0– 48)at thesame time.
Very important:precisepipetting!
Gene t=0 t=12 t=24 t=36
Ct Reference 15 15 15 15
Ct Hox gene 30 28 26 24
ΔCt 15 13 11 9
ΔΔCt 15-13=2(cicli) 15-11=4 15-9=6
2ΔΔCt 22=4 24=16 26=64
0 12 24 36
10203040506070
1
416
64
timefoldexpressio
nchange
of
Hoxg
eneΔΔC t(t=
0)se
t“1”
Basics fortheanalysis ofreal-timePCRdata:Ct andΔCt andΔΔCt
1
Real-Time PCR has become a cornerstone of molecular biology:
• Gene expression analysis• Cancerresearch• Drugresearch
• Disease diagnosis and management• Viralquantification
• Food testing• PercentGMOfood
• Animal and plant breeding• Genecopynumber
WhatisReal-TimePCRusedfor?
Forallapplications thatrequirethequantification ofRNA/DNAsequences